Many agriculturally important crops are susceptible to infection by plant viruses, particularly papaya ringspot virus, which can seriously damage a crop, reduce its economic value to the grower, and increase its cost to the consumer. Attempts to control or prevent infection of a crop by a plant virus such as papaya ringspot virus have been made, yet viral pathogens continue to be a significant problem in agriculture.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous in that the genetic material which provides the protection is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it possesses the ability to suppress or retard the multiplication of a virus, or the development of pathogenic symptoms. "Resistant" is the opposite of "susceptible," and may be divided into: (1) high, (2) moderate, or (3) low resistance, depending upon its effectiveness. Essentially, a resistant plant shows reduced or no symptom expression, and virus multiplication within it is reduced or negligible. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
Potyviruses are a distinct group of plant viruses which are pathogenic to various crops, and which demonstrate cross-infectivity between plant members of different families. Generally, a potyvirus is a single-stranded RNA virus that is surrounded by a repeating protein monomer, which is termed the coat protein (CP). The majority of the potyviruses are transmitted in a nonpersistent manner by aphids. As can be seen from the wide range of crops affected by potyviruses, the host range includes such diverse families of plants as Solanaceae, Chenopodiaceae, Gramineae, Compositae, Leguminosae, Dioscroeaceae, Cucurbitaceae, and Caricaceae. Potyviruses include watermelon mosaic virus II (WMVII); zucchini yellow mosaic virus (ZYMV), potato virus Y, tobacco etch and many others.
Another potyvirus of economic significance is papaya ringspot virus (PRV). Two groups of PRV have been identified: the "P" or "papaya ringspot" type infects papayas; and the "W" or "watermelon" type infects cucurbits, e.g., squash, but it is unable to infect papaya. Thus, these two groups can be distinguished by host range differences.
The potyviruses consist of flexous, filamentous particles of dimensions approximately 780.times.12 nanometers. The viral particles contain a single-stranded positive polarity RNA genome containing about 10,000 nucleotides. Translation of the RNA genome of potyviruses shows that the RNA encodes a single large polyprotein of about 330 kD. This polyprotein contains several proteins; these include the coat protein, nuclear inclusion proteins NIa and NIb, cytoplasmic inclusion protein (CI), and other proteases and movement proteins (see FIG. 1). These proteins are found in the infected plant cell and form the necessary components for viral replication. One of the proteins contained in the polyprotein is a 35 kD capsid or coat protein which coats and protects the viral RNA from degradation. One of the nuclear inclusion proteins, NIb, is an RNA replicase component and is thought to have polymerase activity. CI, a second inclusion protein, is believed to participate in the replicase complex and have a helicase activity. NIa, a third inclusion protein, has a protease activity. In the course of potyvirus infection, NIa and NIb are translationally transported across the nuclear membrane into the nucleus of the infected plant cell at the later stages of infection and accumulate to high levels.
The location of the protease gene appears to be conserved in these viruses. In the tobacco etch virus, the protease cleavage site has been determined to be the dipeptide Gln-Ser, Gln-Gly, or Gln-Ala. Conservation of these dipeptides at the cleavage sites in these viral polyproteins is apparent from the sequences of the above-listed potyviruses.
Expression of the coat protein genes from tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X, among others, in transgenic plants has resulted in plants which are resistant to infection by the respective virus. For reviews, see Fitchen et al., Annu. Rev. Microbiol., 4, 739 (1993) and Wilson, Proc. Natl. Acad. Sci. USA, 90 3134 (1993). For papaya ringspot virus, Ling et al. (Bio/Technology, 9, 752 (1991)) found that transgenic tobacco plants expressing the PRV coat protein (CP) gene isolated from the PRV strain HA 5-1 (mild) showed delayed symptom development and attenuation of symptoms after infection by a number of potyviruses, including tobacco etch (TEV), potato virus Y (PVY), and pepper mottle virus (PeMV). PRV does not infect tobacco, however. Thus, PRV CP transgenic tobacco plants cannot be used to evaluate protection against PRV. Fitch et al. (Bio/Technology, 10 , 1466 (1992)), Gonsalves (American J. of Bot., 79, 88 (1992)), and Lius et al. (91st Annual Meeting of the American Society for Horticultural Science Hortscience, 29, 483 (1994)) reported that four R.sub.o papaya plants made transgenic for a PRV coat protein gene taken from strain HA 5-1 (mild) displayed varying degrees of resistance against PRV infection, and one line (S55-1) appeared completely resistant to PRV. This appears to be the only papaya line that shows complete resistance to PRV infection.
Even though coat protein mediated viral resistance has proven to be useful in a variety of situations, it may not always be the most effective or desirable means for providing viral resistance. In such instances, it would be advantageous to have other methods for conferring viral resistance to plants. Expression of the protease gene (NIa) from tobacco vein mottle virus (TVMV) and potato virus Y (PVY) in transgenic plants has shown the feasibility of using protease gene constructs to produce transgenic plants protected against potyvirus infection (Maiti et al., J. Cell. Biochem., Suppl. 16F, 217(1992); Vardi et al., Proc. Natl. Acad. Sci. U.S.A., 90, 7513 (1993); Maiti et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6110 (1993)). Maiti et al. (1993) showed that the expression of the NIa gene of TVMV in tobacco plants rendered these plants highly resistant to TVMV challenge. In addition, Maiti et al. showed that the NIa gene expressed in these plants was proteolytically active. Vardi et al. transformed tobacco plants with PVY NIa constructs. R.sub.1 progeny from two lines derived from these transformed plants were resistant to challenge with virus.
There is a continuing need for the transgenic expression of genes derived from potyviruses at levels which confer resistance to infection by these viruses.